IL-15 is a cytokine which, like IL-2, has originally been described as a T cell growth factor (1). The two cytokines belong to the four α-helix bundle family, and their membrane receptors share two subunits (the IL-2R/IL-15Rβ and γ chains) responsible for signal transduction (2). The IL-2Rβ/γ complex is an intermediate affinity receptor for both cytokines. It is mainly expressed by most NK cells, and can be activated in vitro by nanomolar concentrations of IL-2 or IL-15.
High affinity IL-2 and IL-15 receptors, which are expressed for example on activated T cells, and which can be activated with picomolar concentrations of either cytokine, contain in addition their own, private, α chain (IL-2Rα and IL-15Rα) that confer cytokine specificity and enhance the affinity of cytokine binding (3).
Both cytokines play pivotal roles in innate and adaptive immunity. Whereas initial in vitro experiments have shown a large functional overlap (induction of the proliferation and cytotoxicity of activated lymphocytes and NK cells, co-stimulation of B cell proliferation and immunoglobin synthesis, chemoattraction of T cells) (1, 4-6), more recent experiments have indicated that the two cytokines exert complementary and even contrasting actions in viva. Whereas IL-2 or IL-2Rα knock out in mice was associated with autoimmune phenotypes with increased populations of activated T and B cells, IL-15 and IL-15Rα knock out resulted in specific defects in NK, NK-T, intraepithelial lymphocytes and memory CD8 T cells (7, 8). Furthermore, IL-2 promotes peripheral tolerance by inducing activation induced cell death (AICD), whereas IL-15 inhibits IL-2 mediated AICD (9), and, unlike IL-2, IL-15 is a survival factor for CD8 memory T cells (10).
In line with these observations, it has been suggested that the major role of IL-2 is to limit continuous expansion of activated T cells, whereas IL-15 is critical for the initiation of T cell division and the survival of memory T cells (11).
A novel mechanism of IL-15 transpresentation has been described, in which IL-15 and IL-15Rα are coordinately expressed by antigen-presenting cells (monocytes, dendritic cells), and IL-15 bound to IL-15Rα is presented in trans to neighboring NK or CD8 T cells expressing only the IL-15Rβ/γ receptor (12). IL-15 transpresentation as a co-stimulatory event occurring at the immunological synapse, now appears to be a dominant mechanism for IL-15 action in vivo (13, 14). It is suggested to play a major role in tumor immunosurveillance (15).
The IL-15Rα and IL-2Rα subunits form a sub-family of cytokine receptors in that they comprise at their N-terminal extracellular parts so called “sushi” structural domains (one in IL-15Rα, two in IL-2Rα) also found in complement or adhesion molecules (16). In both cases, these sushi domains have been shown to bear most of the structural elements responsible for the cytokine binding.
Whereas IL-2Rα alone is a low affinity receptor for IL-2 (Kd=10 nM), IL-15Rα binds IL-15 with high affinity (Kd=100 pM), Shedding of IL-2Rα by proteolysis is a natural mechanism that participates in the down regulation of lymphocyte activation. IL-2Rα is cleaved by Der p1, a major mite allergen, to inhibit Th1 cells and favor an allergic environment (17), and by tumor-derived metalloproteinases to suppress the proliferation of cancer-encountered T cells (18). The soluble IL-2Rα thus generated is a competitive inhibitor of IL-2 action in vitro. However, it remains a low affinity IL-2 binder, and it is not likely to efficiently participate in down regulation of IL-2 activity in vivo.
It has been recently shown that a soluble form of the human IL-15Rα can also be naturally released from IL-15Rα positive cells by a shedding process involving MMPs (19). In contrast to soluble IL-2Rα, this soluble IL-15Rα receptor was able to bind IL-15 with high affinity, and efficiently blocks proliferation driven through the high affinity IL-15Rα/β/γ signaling complex. This result was consistent with the concept of sIL-15Rα behaving, like its homolog sIL-2Rα, as an antagonist, and with inhibitory effects of mouse sIL-15Rα in vitro or in vivo (20, 21).
Here, the present inventors show that a fragment essentially consisting of the sushi domain of IL-15Ralpha (=IL-15Rα) has an opposite action. Such a fragment is able to enhance the binding, as well as the bioactivity of IL-15 through the IL-15Rbeta/gamma (=IL-15Rβ/γ) intermediate affinity receptor, without affecting those through the high affinity receptor. In addition, the present inventors describe fusion proteins which behave as potent super-agonists of the IL-15Rβ/γ complex. Such fusion proteins comprise an IL-15Rbeta/gamma binding entity, such as IL-15 (or a conservative fragment, agonist, mimetic thereof), fused by covalence e.g., by a flexible linker, to IL-15Rα or to an IL-15Ralpha fragment which has retained the sushi domain of IL-15Ralpha.
To the best of the inventors' knowledge, there is only one prior art which reports a stimulating effect for a compound comprising an IL-15Ralpha-related element, namely the commercially available form of soluble IL-15Ralpha.
It is the Giron-Michel et al. publication, which is entitled “Membrane-bound and soluble IL-15/IL-15Ralpha complexes display differential signalling and functions on human haematopoietic progenitors” (Blood, 1 Oct. 2005, Vol. 106, No. 7, pp. 2302-2310; pre-published online in June 2005).
The Giron-Michel et al. publication discloses that (see FIG. 7 of Giron-Michel et al.):                recombinant IL-15 (rIL-15) induces a significant anti-apoptotic effect when it is used at a dose of 10 ng/ml, and that        rIL-15 does not induce any significant anti-apoptotic effect at a dose of 0.1 ng/mL, but that        rIL-15 at a dose of 0.1 ng/mL induces a significant anti-apoptotic effect when it is used with the commercially available form of soluble IL-15Ralpha.        
The soluble IL-15Ralpha that is used by Giron-Michel et al. is the commercially available form of IL-15Ralpha (available from R&D Systems, under reference 147-IR). This soluble IL-15Ralpha is a modified form of soluble IL-15Ralpha, which lacks exon 3. The form of soluble IL-15Ralpha that is used by Giron-Michel et al. hence comprises the exon 2 encoded part of IL-15Ralpha, directly linked to the exon 4 encoded part of IL-15Ralpha, without comprising any exon 3 encoded part of IL-15Ralpha. The form of soluble IL-15Ralpha that is used by Giron-Michel et al. does therefore not correspond to a fragment of IL-15Ralpha, but to a modified form thereof.
The form of soluble IL-15Ralpha that is used by Giron-Michel et al. further comprises a Fc fragment (human IgG), linked thereto by covalence. A Fc fragment does not bind to IL-15Rbeta/gamma. The Giron-Michel et al. publication does therefore not disclose any compound wherein the soluble IL-15Ralpha form would be linked by covalence to an IL-15Rbeta/gamma binding entity.
The Giron-Michel et al. publication further discloses an anti-apoptotic effect, but does not disclose any effect on the proliferation and/or activation of IL-15Rbeta/gamma-positive cells. It can further be noted that the disclosed anti-apoptotic effect assay does not comprise any control samples which would contain (i) the soluble IL-15Ralpha-Fc fragment in the absence of rIL-15 or (ii) a soluble IL-15Ralpha without any Fc fragment. The disclosed anti-apoptotic effect therefore cannot be directly attributed to the IL-15Ralpha part of the compound that is used.
The Giron-Michel et al. publication does not further contain any hint to the sushi domain of IL-15Ralpha (nor to the hinge region that is absent from the soluble IL-15Ralpha form that is being used in this prior art), nor does it contain a hint to the IL-15beta/gamma signalling pathway.
The present invention describes for the first time the structural units which are necessary to, and especially advantageous for, the induction and/or stimulation of an IL-15 biological action, the specific triggering of the IL-15beta/gamma signalling pathways, and the induction and/or stimulation of the proliferation of NK and/or T cells. The present invention thereby represents a technical contribution over the prior art, which enables previously-unattained biological and medical applications.
It is therefore believed that, when analysed on an a priori basis, the Giron-Michel et al. publication does not teach the claimed invention to the person of ordinary skill in the art, and does not guide the skilled person to the claimed invention.